This invention relates to methods for detecting the presence of unknown pathogens or microorganisms and, more particularly, to in vitro methods for detecting the presence of unknown pathogens in a more rapid manner and through the employment of procedures which can be readily and reliably carried out in clinical microbiology laboratories.
A critical factor in the clinical management of infectious diseases lies in the establishment of the identity of the etiologic agent or pathogen responsible for the infection. In most instances, the identification of the infecting microbe is central in making decisions respecting the appropriate therapy to be utilized. In this regard, the attending physician necessarily places heavy reliance upon the clinical microbiology laboratory to provide the essential data required to initiate a rational regimen of treatment.
Factors comprising procedures currently used in detecting pathogens in clinical specimens include the time required for detection, inability to rapidly cultivate the infectious agent in all instances, and difficulties concerning cross reactivity of reagents when performing assays directly from clinical specimens or samples.
Conventionally, the primary basis for the identification of pathogenic bacteria involves the detection of a products resulting from the metabolism of the unknown organism (Lennette et al., Manual of Clinical Microbiology, 1974, Amer. Soc. for Microbiol.), e.g. the detection of various enzymatic capabilities of a microbe by identification of specific substrate utilization or formation of specific metabolic end products. In most instances, identity of the unknown organism is based on color changes in multiple separate reactions (as many as 20-30) in order to arrive at a species level identification. In all cases, considerable growth of the microorganism under defined conditions is required to permit accumulation of the substances to be detected. The tests employed heretofore undesirably require numerous reagents and media, are timeconsuming and also require a considerable degree of skill and training on the part of clinical laboratory personnel.
During the past twenty years or so, various systems have been devised in an effort to simplify and shorten the time involved in the process of microorganism identification (Nord et al., Med Microbiol. Immunol., 1974, 159:211-220; Sakazaki, Media Circle, 1975, 20:227-235; and Robertson et al., J. Clin. Microbiol., 1976, 3:421-424). However, these systems basically represent variations on the same theme and rely on the same basic principle of metabolic product detection.
Reference may be made to the following literature publications as being representative of the state of the art as regards the rapid identification of infectious agents or microorganisms. Totten et al., DNA Hybridization Technique for the Detection of Neisseria gonorrhaeae in Men with Urethritis, Abstracts, Amer. Soc. Microbiol., March, 1982, discloses a technique for detecting Neisseria gonorrhaeae in patient specimens using a modification of the DNA hybridization method with the gonococcal cryptic plasmid as the radiolabelled probe. Moseley et al., Identification of Enterotoxigenic Escherichia coli by Colony Hybridization Using Three Enterotoxin Gene Probes, J. Infect. Dis. 145:863-869, discloses the applicability and limitation of the DNA hybridization technique for identifying enterotoxigenic Escherichia coli (ETEC).
A series of abstracts (C87 to C95, Abstracts of Annual Meeting-1982, Amer. Soc. Microbiol.) discloses evaluations and comparisons of multi-tube metabolic product test systems (API). Other abstracts in this series (C120 to C123 and C125) disclose evaluations of an automated radiometric blood culture system ("Bactec", Johnston Laboratories, Inc.) which relies upon the detection of metabolic products. Still other abstracts in this series (C124 to C128) describe blood culture systems for the concentration and separation of bacteria from blood samples. Abstract C254 in this same series describes a two-step procedure for expediting the recovery of microorganisms from blood.
Recently, it has become possible to produce large amounts of DNA fragments specific for an etiologic agent utilizing recombinant DNA molecules. Once the specificity of any particular fragment(s) for a certain pathogen has been established empirically, this DNA fragment or probe can be used to detect the presence of that pathogen through the probe's ability to hybridize with its complementary nucleic acid sequence of the pathogen, i.e. through DNA-DNA hybridization or DNA-RNA hybridization in the case of infectious viral entities that contain RNA genomes. RNA probes may also be used.
The use of such DNA probes in a diagnostic method for the detection of pathogens is disclosed in Falkow et al. U.S. Pat. No. 4,358,535. This patent generally discloses a method in which clinical isolates are cultivated, expanding the number of microorganisms, the resulting colonies are lysed and the genome is denatured and then fixed. Alternatively, clinical samples containing the pathogen are deposited or spotted onto an inert support. The sample is then treated and lysed to liberate the DNA from microbes present in the sample and to fix the DNA in substantially single stranded form at the same site on the support where the sample was deposited. Subsequently, the single stranded DNA on the support is contacted with a labeled polynucleotide probe with a nucleotide sequence complementary to that of the pathogen under hybridizing conditions whereby hybridization of the probe to the single stranded DNA of the pathogen is diagnostic of the presence of the pathogen. The patent states that the lysis conditions are devised such that the cells or colonies do not migrate and their DNA remains affixed in place on the surface of the support where they were situated. The method disclosed requires several days for detection of the pathogen involved.
In a more recent paper by Falkow and others (Totten et al., J. Infect. Dis., 1983, 148:462), the DNA hybridization technique was applied to the detection of Neisseria gonorrhaeae in men with urethritis directly from clinical specimens. However, the technique requires at least three days of reaction time to detect bacteria directly in patient specimens and thus is not immediately applicable to the clinical laboratory.
Brautigam et al. (J. Clin. Microbiol., 1980, 12:226-234) describe a method of typing clinical isolates of herpes simplex virus using hybridization between unlabeled DNA from infected cultures and tritium-labeled virus DNA. While the procedure can be completed within a day, it requires expensive, elaborate equipment and is not readily adapted to the processing of several clinical samples at any one time. It also requires performance by one relatively experienced in nucleic acid chemistry.
Moseley et al. (J. Infect. Dis., 1980, 142:892-898) disclose a method for detecting large numbers of isolates of enterotoxigenic Escherichia coli in which radiolabeled fragments of DNA encoding the heat-labile or heat-stable toxins were used as hybridization probes for homologous DNA sequences in E. coli colonies grown and lysed in situ on nitrocellulose filters. This method is also time-consuming and requires several days for completion.
Brechot et al. (The Lancet, October 10, 1981, p. 765-767) describe the use of DNA-DNA hybridization in the detection of hepatitis B virus in liver and serum. The procedure described requires tissue biopsies, followed by DNA extraction and subsequent steps which take about 3 to 4 days to complete.
Berninger et al. (J. Med. Virol., 1982, 9:57-68) discloses an assay based on nucleic acid hybridization which detects and quantitates hepatitis B virus (HBV) DNA in particles present in serum. The assay employs the complete hepatitis B virus DNA as a probe and the times required to complete the assay are relatively lengthy.
Thus, while the principle of nucleic acid hybridization has been recognized and used in the detection of pathogens, a need continues to exist for a more rapid, practical and accurate method for detecting pathogens directly from clinical samples to enhance the usefulness of the service rendered by clinical microbiology laboratories.